Interest in the identification and detection of small RNAs has expanded rapidly in the last few years, particularly with the recent discoveries related to microRNAs and small interfering RNAs (siRNA), both of which have a powerful affect on the expression of genes. siRNA molecules, which are generally short, double stranded RNA, are used to silence the expression of specific genes at the post-transcriptional level by a pathway known as RNA interference (RNAi). microRNAs, small regulatory RNA molecules, have been shown to regulate target gene expression in various organisms. siRNA and microRNA molecules generally range between about 15 and 30 nucleotides in length. Other types of small RNAs include small nuclear RNAs (snRNAs) and small nucleolar RNAs (snoRNAs), both of which are involved in mRNA and rRNA processing, as well as tRNAs (about 70-90 bases), and 5S rRNA (about 120 bases), which are both involved in protein translation.
A number of nucleic acid detection technologies, such as INVADER assays and TAQMAN assays, generally require a certain “footprint” for various probes and primers to sit down onto a target nucleic acid in order to detect the target nucleic acid or a specific target nucleotide in the target nucleic acid. This can be problematic if the target nucleic acid itself if very short (e.g., small RNAs), or if the nucleotide to be detected is close to the 5′ or 3′ end of the target nucleic acid.
What is needed, therefore, are methods, compositions, and kits that allow nucleic acid assays that generally require a “footprint,” to be used for detecting targets and target nucleotides when the required footprint is not present in the original target sequence.